Method of solubilizing and encapsulating itraconazole

ABSTRACT

Methods for preparing microspheres containing imidazole derivatives are provided. Also provided is the use of imidazole derivatives containing microspheres for treating fungal infections. Oral dosage forms for oral administration are also provided.

This is a divisional of U.S. application Ser. No. 08/967,486, filed Nov. 11, 1997, now U.S. Pat. No. 6,100,285, which is a divisional of U.S. application Ser. No. 08/475,887, filed Jun. 7, 1995, now U.S. Pat. No. 5,750,147. These prior applications are hereby incorporated herein by reference, in their entirety.

FIELD OF THE INVENTION

The present invention relates to the preparation of solutions containing imidazole derivatives and to the use of those solutions in the preparation of microspheres. The imidazole derivative containing microspheres are effective in treating fungal infections, particularly in mammals. The microspheres facilitate the oral administration of relatively large amounts of the imidazole derivative, with increased bioavailability.

BACKGROUND OF THE INVENTION

Many present systems for delivering active agents to targets are severely limited by biological, chemical, and physical barriers, which are imposed by the environment through which delivery occurs, the environment of the target itself, or the target itself. Delivery is also limited, in many instances, by the chemical nature of the active agent. For example, oral delivery is generally ineffective with active agents that are poorly water-soluble.

The imidazole derivative family of compounds is particularly effective against a broad range of fungal infections such as those caused by Trichophyton rubrum, Tricophyton mentagrophytes, Epidermophyton floccsum, and Candida albicans, but these compounds are either partially water soluble or insoluble in water. For example, the solubility of itraconazole in water is less than 0.00001 g/ml.

Partially because imidazole derivatives are typically insoluble in water, they are difficult to administer orally. Consequently although imidazole derivatives are frequently prescribed for the treatment of fungal infections, they have been available only in topical preparations or in oral formulations with limited bioavailability.

In recent years, fungal infections, such as those caused by Candida albicans in particular have become more prevalent and intractable due to their appearance in immunocompromised patients, such as those infected with Human Immunodeficiency Virus (HIV) or those suffering from Acquired Immunodeficiency Syndrome (AIDS).

For example, U.S. Pat. No. 3,717,655 discloses imidazole derivatives which have antifungal and antibacterial activity. These compounds are almost insoluble in aqueous solutions such as water and are very poorly soluble in polar solvents such as ethanol.

Das et al., U.S. Pat. No. 4,912,124, disclose a solvent system for imidazole derivatives that include mixtures of a polar solvent, a polyhydric alcohol that acts as a solubilizing agent, a nonionic or amphoteric surfactant, and a cosmetic humectant. Solutions containing at least 1 percent by weight of the imidazole derivatives can be formulated using this solvent system. However, these formulations are suitable for external topical use only.

Accordingly, there is a need for orally deliverable forms of imidazole derivative antifungal agents.

SUMMARY OF THE INVENTION

The present invention provides solutions comprising:

(a) at least about 2.5 parts by weight, based upon 100 parts by weight of solution, of a solute having the formula

wherein R, R¹, and R² are independently hydrogen or lower alkyl;

R³ is hydrogen, methyl or ethyl;

R⁴ is hydrogen or methyl

Ar is phenyl, monohalophenyl, dihalophenyl, trihalophenyl, mono(lower alkyl)phenyl, di(lower alkyl)phenyl, lower alkoxyphenyl, or halothienyl;

Ar¹ is phenyl, monohalophenyl, dihalophenyl, trihalophenyl, mono(lower alkyl)phenyl, di(lower alkyl)phenyl, lower alkoxyphenyl, or cyanophenyl; and

n is 1 or 2; and

(b) a solubilizing effective amount of a solvent comprising at least one volatile organic acid solvent.

Imidazole derivative microspheres are also provided. These microspheres comprise:

(a) an imidazole derivative active agent having the formula

 wherein

R, R¹, and R² are independently hydrogen or lower alkyl;

R³ is hydrogen, methyl or ethyl;

R⁴ is hydrogen or methyl

Ar is phenyl, monohalophenyl, dihalophenyl, trihalophenyl, mono(lower alkyl)phenyl, di(lower alkyl)phenyl, lower alkoxyphenyl, or halothienyl;

Ar¹ is phenyl, monohalophenyl, dihalophenyl, trihalophenyl, mono (lower alkyl)phenyl, di(lower alkyl)phenyl, lower alkoxyphenyl, or cyanophenyl; and

n is 1 or 2; and

(b) a microsphere forming carrier selected from the group consisting of

(i) a proteinoid;

(ii) an acylated amino acid, poly amino acid, or a salt thereof;

(iii) an sulfonated amino acid, poly amino acid, or a salt thereof;

(iv) a protein or a salt thereof;

(v) an enteric coating material; or

(vi) any combination thereof.

Also contemplated by the present invention is a method for preparing these microspheres. The method comprises:

(A) nebulizing a solution comprising

(a) an imidazole active agent having the formula

 wherein

R, R¹, and R² are independently hydrogen or lower alkyl;

R³ is hydrogen, methyl or ethyl;

R⁴ is hydrogen or methyl

Ar is phenyl, monohalophenyl, dihalophenyl, trihalophenyl, mono(lower alkyl)phenyl, di(lower alkyl)phenyl,. lower alkoxyphenyl, or halothienyl;

Ar¹ is phenyl, monohalophenyl, dihalophenyl, trihalophenyl, mono(lower alkyl)phenyl, di(lower alkyl)phenyl, lower alkoxyphenyl, or cyanophenyl; and

n is 1 or 2;

(b) an active agent and carrier solubilizing effective amount of a solvent comprising an aqueous solution of at least one volatile organic solvent; and wherein the volume:volume ratio of acid to water in said carrier solution is at least about 3:7, and

(c) microsphere forming a carrier selected from the group consisting of

(i) a proteinoid;

(ii) an acylated amino acid or poly amino acid or a salt thereof;

(iii) an sulfonated amino acid or poly amino acid or a salt thereof;

(iv) a protein or a salt thereof;

(v) an enteric coating material, or

(vi) any combination thereof; and

(B) decreasing said ratio to less than about 3:7, to yield said microspheres. Alternatively, the active agent and the carrier can be solubilized in separate solutions. The separate solutions can be nebulized together and the acid to water ratio then decreased as above.

Methods for the oral administration of imidazole derivatives are also contemplated wherein the microsphere compositions above are orally administered to an animal in need of this treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a scanning electron micrograph (SEM) taken at a magnification of 2,000× of microspheres containing itraconazole prepared according to the present invention.

FIG. 1B is a SEM taken at a magnification of 15,000× of microspheres containing itraconazole prepared according to the present invention.

FIG. 1C is a SEM taken a magnification of 20,000× of microspheres containing itraconazole prepared according to the present invention.

FIG. 1D is a SEM taken at a magnification of 20,000× of microspheres containing itraconazole prepared according to the present invention.

FIG. 1E is a SEM taken at a magnification of 20,000× of microsphers containing itraconazole prepared according to the present invention.

FIG. 1F is a SEM taken at a magnification of 20,000× of microsphers containing itraconazole prepared according to the present invention.

FIG. 1G is a SEM taken at a magnification of 2,000× of microsphers containing itraconazole prepared according to the present invention, after being mechanically crused.

FIG. 1H is a SEM taken at a magnification of 3,500× of microsphers containing itraconazole prepared according to the present invention, after being mechanically crused.

DETAILED DESCRIPTION OF THE INVENTION

It has now been discovered that water insoluble or partially soluble imidazole derivatives can be solubilized in volatile organic acids. The resultant solutions can be used to prepare imidazole containing microspheres which are suitable for oral administration to animals.

Imidazole Derivatives

The active agents of the present invention are imidazole derivatives having the formula:

wherein

R, R¹ and R² are independently hydrogen or lower alkyl;

R³ is hydrogen, methyl or ethyl;

R⁴ is hydrogen or methyl

Ar is phenyl, monohalophenyl, dihalophenyl, trihalophenyl, mono (lower alkyl)phenyl, di(lower alkyl)phenyl, lower alkoxyphenyl, or halothienyl;

Ar¹ is phenyl, monohalophenyl, dihalophenyl, trihalophenyl, mono(lower alkyl)phenyl, di(lower alkyl)phenyl, lower alkoxyphenyl, or cyanophenyl; and

n is 1 or 2.

A preferred imidazole derivative is itraconazole. Itraconazole is a synthetic triazole imidazole derivative 1:1:1:1 racemic mixture of four diastereomers (two enantiomeric pairs), each possessing three chiral centers (Physicians Desk Reference 48th Ed., pg. 1097, 1994).

Imidazole Derivative Solutions

The solutions prepared in accordance with the present invention allow for the solubilization of imidazole derivatives at concentrations suitable for processing into orally administrable forms having acceptable bioavailability.

In accordance with the present invention, imidazole derivatives are solubilized in volatile organic acid solvent(s). Preferred acid solvents for the imidazole derivatives are acetic acid and formic acid. Preferably, the solvent itself is an aqueous solution of the acid. Most preferably the volume:volume ratio of the acid to the total volume of the solvent is 3:7 or greater. It has been found that by using this solvent system up to a 50% solution of imidazole derivative can be prepared.

Dissolution is achieved by simple mixing, with heating if necessary. The more concentrated the acid in the solvent, the greater the amount of active agent that can be incorporated into the solution. If lower concentrations of acid are required for the end use of the solution, the active agent can first be dissolved in a more concentrated acid solution, and the resultant solution then slowly diluted further, preferably with water.

Preferably, the solution comprises from about 3 to about 40 percent by weight of solute and from about 60 to about 97 parts by weight of solvent based upon 100 parts by weight of solution.

The solvent itself, preferably comprises from about 30 to about 80 parts by volume of acid and from about 70 to about 20 parts by volume of water based upon 100 parts by volume of solvent. Most preferably, the solvent comprises from about 40 to about 50 parts by volume of acid and from about 60 to about 50 parts by volume of water based upon 100 parts by volume of solvent.

Microspheres

Microspheres are useful in the delivery of active agents because they protect an active agent cargo until it is delivered to a target. Microsphers are particularly useful in the oral delivery of biologically active agents such as for example, pharamaceutically active agents.

Microspheres containing an active agent can be generally of the matrix form or the capsule form. In a hollow matrix spheroid form, the center of the sphere is hollow and the cargo or active agent is distributed throughout a carrier matrix. In a solid matrix form, the carrier matrix forms a continuum in which the cargo is distributed. In the microcapsule form, the encapsulated material or cargo can be either in solution or a solid, with the carrier forming a shell around the cargo.

The methods of the present invention are cost-effective for preparing microspheres which contain imidazole derivatives, are simple to perform, and are amenable to industrial scale-up for commercial production.

Carriers

Carriers suitable for use in the present invention are microsphere forming carriers. These carriers include, without limitation, proteinoids; acylated amino acids, poly amino acids or salts thereof; sulfonated amino acids, poly amino acids or salts thereof; proteins or salts thereof, enteric coating materials; or any combination thereof.

Amino acids are the basic materials used to prepare many of the carriers useful in the present invention. Amino acids include any carboxylic acid having at least one free amino group and include naturally occurring and synthetic amino acids. The preferred amino acids for use in the present invention are ∝-amino acids and, most preferably, are naturally occurring ∝-amino acids. Many amino acids and amino acid esters are readily available from a number of commercial sources such as Aldrich Chemical Co. (Milwaukee, Wis., USA); Sigma Chemical Co. (St. Louis, Mo., USA); and Fluka Chemical Corp. (Ronkonkoma, N.Y., USA).

Representative, but not limiting, amino acids suitable for use in the present invention are generally of the formula

wherein:

R⁵ is hydrogen, C₁-C₄ alkyl, or C₂-C₄ alkenyl;

R⁶ is C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, phenyl, naphthyl, (C₁-C₁₀ alkyl) phenyl, (C₂-C₁₀ alkenyl) phenyl, (C₁-C₁₀ alkyl) naphthyl, (C₂-C₁₀ alkenyl) naphthyl, phenyl (C₁-C₁₀ alkyl), phenyl (C₂-C₁₀ alkenyl), naphthyl (C₁-C₁₀ alkyl), or naphthyl (C₂-C₁₀ alkenyl);

R⁶ being optionally substituted with C₁-C₄ alkyl, C₂-C₄ alkenyl, C₁C₄ alkoxy, —OH, —SH, —CO₂R⁷, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, heterocycle having 3-10 ring atoms wherein the hetero atom is one or more of N, O, S, or any combination thereof, aryl, (C₁-C₁₀ alk)aryl, ar(C₁-C₁₀ alkyl) or any combination thereof;

R⁶ being optionally interrupted by oxygen, nitrogen, sulfur, or any combination thereof; and

R⁷ is hydrogen, C₁-C₄ alkyl, or C₂-C₄ alkenyl.

The preferred naturally occurring amino acids for use in the present invention as amino acids or components of a peptide are alanine, arginine, asparagine, aspartic acid, citrulline cysteine, cystine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, hydroxyproline, β-carboxyglutamic acid, γ-carboxyglutamic acid, phenylglycine, or O-phosphoserine. The most preferred amino acids are arginine, leucine, lysine, phenylalanine, tyrosine, tryptophan, valine, and phenylglycine.

The preferred non-naturally occurring amino acids for use in the present invention are β-alanine, α-amino butyric acid, γ-amino butyric acid, γ-(aminophenyl) butyric acid, α-amino isobutyric acid, ε-amino caproic acid, 7-amino heptanoic acid, β-aspartic acid, aminobenzoic acid, aminophenyl acetic acid, aminophenyl butyric acid, γ-glutamic acid, cysteine (ACM), ε-lysine, methionine sulfone, norleucine, norvaline, ornithine, d-ornithine, p-nitro-phenylalanine, hydroxy proline, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, and thioproline.

Poly amino acids are either peptides or two or more amino acids linked by a bond formed by other groups which can be linked, e.g., an ester or an anhydride linkage. Special mention is made of non-naturally occurring poly amino acids and particularly non-naturally occurring hetero-poly amino acids, i.e. of mixed amino acids.

Peptides are two or more amino acids joined by a peptide bond. Peptides can vary in length from di-peptides with two amino acids to polypeptides with several hundred amino acids. See, Walker, Chambers Biological Dictionary, Cambridge, England: Chambers Cambridge, 1989, page 215. Special mention is made of non-naturally occurring peptides and particularly non-naturally occurring peptides of mixed amino acids. Special mention is also made of di-peptides tri-peptides, tetra-peptides, and penta-peptides, and particularly, the preferred peptides are di-peptides and tri-peptides. Peptides can be homo- or hetero- peptides and can include natural amino acids, synthetic amino acids, or any combination thereof.

Proteinoids

Proteinoids are artificial polymers of amino acids. Proteinoids preferably are prepared from mixtures of amino acids. Preferred proteinoids are condensation polymers, and most preferably, are thermal condensation polymers. These polymers may be directed or random polymers. Proteinoids can be linear, branched, or cyclical, and certain proteinoids can be units of other linear, branched, or cyclical proteinoids.

Special mention is made of diketopiperazines. Diketopiperazines are six member ring compounds. The ring includes two nitrogen atoms and is substituted at two carbons with two oxygen atoms. Preferably, the carbonyl groups are at the 2 and 5 ring positions. These rings can be optionally, and most often are, further substituted.

Diketopiperazine ring systems may be generated during thermal polymerization or condensation of amino acids or amino acid derivatives. (Gyore, J; Ecet M. Proceedings Fourth ICTA (Thermal Analysis), 1974, 2, 387-394 (1974)). These six membered ring systems were presumably generated by intra-molecular cyclization of the dimer prior to further chain growth or directly from a linear peptide (Reddy, A. V., Int. J. Peptide Protein Res., 40, 472-476 (1992); Mazurov, A. A. et al., Int. J. Peptide Protein Res., 42, 14-19 (1993)).

Diketopiperazines can also be formed by cyclodimerization of amino acid ester derivatives as described by Katchalski et al., J. Amer. Chem. Soc., 68, 879-880 (1946), by cyclization of dipeptide ester derivatives, or by thermal dehydration of amino acid derivatives and high boiling solvents as described by Kopple et al., J. Org. Chem., 33 (2), 862-864 (1968).

Diketopiperazines typically are formed from α-amino acids. Preferably, the α-amino acids of which the diketopiperazines are derived are glutamic acid, aspartic acid, tyrosine, phenylalanine, and optical isomers of any of the foregoing.

Modified Amino Acids and Poly Amino Acids

Modified amino acids, poly amino acids, or peptides are either acylated or sulfonated and include amino acid amides and sulfonamides.

Acylated Amino Acids and Poly Amino Acids

Although any acylated amino acids or poly amino acids are useful in the present invention, special mention is made of acylated amino acids having the formula

Ar² —Y—(R⁸)_(n)—OH  III

wherein Ar² is a substituted or unsubstituted phenyl or naphthyl;

R⁸ has the formula

wherein:

R⁹ is C₁ to C₂₄ alkyl, C₁ to C₂₄ alkenyl, phenyl, naphthyl, (C₁ to C₁₀ alkyl) phenyl, (C₁ to C₁₀ alkenyl) phenyl, (C₁ to C₁₀ alkyl) naphthyl, (C₁ to C₁₀ alkenyl) naphthyl, phenyl (C₁ to C₁₀ alkyl), phenyl (C₁ to C₁₀ alkenyl), naphthyl (C₁ to C₁₀ alkyl) and naphthyl (C₁ to C₁₀ alkenyl);

R⁹ is optionally substituted with C₁ to C₄ alkyl, C₁ to C₄ alkenyl, C₁ to C₄ alkoxy, —OH, —SH and —CO₂R¹¹, cycloalkyl, cycloalkenyl, heterocyclic alkyl, alkaryl, heteroaryl, heteroalkaryl, or any combination thereof;

R¹¹ is hydrogen, C₁ to C₄ alkyl or C₁ to C₄ alkenyl;

R⁹ is optionally interrupted by oxygen, nitrogen, sulfur or any combination thereof; and

R¹⁰ is hydrogen, C₁ to C₄ alkyl or C₁ to C₄ alkenyl.

Special mention is also made of those having the formula

wherein:

R¹² is

(i) C₃-C₁₀ cycloalkyl, optionally substituted with C₁-C₇ alkyl, C₂-C₇ alkenyl, C₁-C₇ alkoxy, hydroxy, phenyl, phenoxy or —CO₂R¹⁵, wherein R¹⁵ is hydrogen, C₁-C₄ alkyl, or C₂-C₄ alkenyl; or

(ii) C₁-C₆ . alkyl substituted with C₃-C₁₀ cycloalkyl;

R¹³ is hydrogen, C₁-C₄ alkyl, or C₂-C₄ alkenyl;

R¹⁴ is C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, phenyl, naphthyl, (C₁-C₁₀ alkyl) phenyl, (C₂-C₁₀ alkenyl) phenyl, (C₁-C₁₀ alkyl) naphthyl, (C₂-C₁₀ alkenyl) naphthyl, phenyl (C₁-C₁₀ alkyl), phenyl (C₂-C₁₀ alkenyl), naphthyl (C₁-C₁₀ alkyl) or naphthyl (C₂-C₁₀ alkenyl);

R¹⁴ being optionally substituted with C₁-C₄ alkyl, C₂-C₄ alkenyl, C₁-C₄ alkoxy, —OH, —SH, —CO₂R¹⁶, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, heterocycle having 3-10 ring atoms wherein the hetero atom is one or more of N, O, S or any combination thereof, aryl, (C₁-C₁₀ alk)aryl, ar(C₁-C₁₀ alkyl), or any combination thereof;

R¹⁴ being optionally interrupted by oxygen, nitrogen, sulfur, or any combination thereof; and

R¹⁶ is hydrogen, C₁-C₄ alkyl, or C₂-C₄ alkenyl.

Acylated amino acids may be prepared by reacting single amino acids, mixtures of two or more amino acids, or amino acid esters with an amine modifying agent which reacts with free amino moieties present in the amino acids to form amides.

Suitable, but non-limiting, examples of acylating agents useful in preparing acylated amino acids include acid chloride acylating agents having the formula

wherein:

R¹⁷ an appropriate group for the modified amino acid being prepared, such as, but not limited to, alkyl, alkenyl, cycloalkyl, or aromatic, and particularly methyl, ethyl, cyclohexyl, cyclophenyl, phenyl, or benzyl, and X is a leaving group. Typical leaving groups include, but are not limited to, halogens such as chlorine, bromine and iodine.

Examples of the acylating agents include, but are not limited to, acyl halides including, but not limited to, acetyl chloride, propyl chloride, cyclohexanoyl chloride, cyclopentanoyl chloride, and cycloheptanoyl chloride, benzoyl chloride, hippuryl chloride and the like; and anhydrides, such as acetic anhydride, propyl anhydride, cyclohexanoic anhydride, benzoic anhydride, hippuric anhydride and the like. Preferred acylating agents include benzoyl chloride, hippuryl chloride, acetyl chloride, cyclohexanoyl chloride, cyclopentanoyl chloride, and cycloheptanoyl chloride.

The amine groups can also be modified by the reaction of a carboxylic acid with coupling agents such as the carbodiimide derivatives of amino acids, particularly hydrophilic amino acids such as phenylalanine, tryptophan, and tyrosine. Further examples include dicyclohexylcarbodiimide and the like.

If the amino acid is multifunctional, i.e. has more than one —OH, —NH₂ or —SH group, then it may optionally be acylated at one or more functional groups to form, for example, an ester, amide, or thioester linkage.

In acylated poly amino acids, one or more of the amino acids may be modified (acylated). Modified poly amino acids may include one or more acylated amino acid(s). Although linear modified poly amino acids will generally include only one acylated amino acid, other poly amino acid configurations can include more than one acylated amino acid. Poly amino acids can be polymerized with the acylated amino acid(s) or can be acylated after polymerization.

Sulfonated Amino Acids and Poly Amino Acids

Sulfonated amino acids and poly amino acids are modified by sulfonating at least one free amine group with a sulfonating agent which reacts with at least one of the free amine groups present.

Special mention is made of compounds of the formula

Ar³—Y—(R¹⁸)_(n)—OH  V

wherein

Ar³ is a substituted or unsubstituted phenyl or naphthyl;

Y is —SO₂—, R¹⁸ has the formula

 wherein:

R¹⁹ is C₁ to C₂₄ alkyl, C₁ to C₂₄ alkenyl, phenyl, naphthyl, (C₁ to C₁₀ alkyl) phenyl, (C₁ to C₁₀ alkenyl) phenyl, (C₁ to C₁₀ alkyl) naphthyl, (C₁ to C₁₀ alkenyl) naphthyl, phenyl (C₁ to C₁₀ alkyl), phenyl (C₁ to C₁₀ alkenyl), naphthyl (C₁ to C₁₀ alkyl) and naphthyl (C₁ to C₁₀ alkenyl);

R¹⁹ is optionally substituted with C₁ to C₄ alkyl, C₁ to C₄ alkenyl, C₁ to C₄ alkoxy, —OH, —SH and —CO₂R²¹ or any combination thereof;

R²¹ is hydrogen, C₁ to C₄ alkyl or C₁ to C₄ alkenyl;

R¹⁹ is optionally interrupted by oxygen, nitrogen, sulfur or any combination thereof; and

R²⁰ is hydrogen, C₁ to C₄ alkyl or C₁ to C₄ alkenyl.

Suitable, but non-limiting, examples of sulfonating agents useful in preparing sulfonated amino acids include sulfonating agents having the formula R²²—SO₂—X wherein R²² is an appropriate group for the modified amino acid being prepared such as, but not limited to, alkyl, alkenyl, cycloalkyl, or aromatics and X is a leaving group as described above. One example of a sulfonating agent is benzene sulfonyl chloride.

Modified poly amino acids and peptides may include one or more sulfonated amino acid(s). Although linear modified poly amino acids and peptides used generally include only one sulfonated amino acid, other poly amino acid and peptide configurations can include more than one sulfonated amino acid. Poly amino acids and peptides can be polymerized with the sulfonated amino acid(s) or can be sulfonated after polymerization.

Proteins

Proteins are naturally occurring (i.e. not artificial) polymers of amino acids.

Enteric Coating Materials

Enteric coating materials known to those skilled in the art such as, for example, cellulose acetate trimellitate (CAT) and cellulose acetate phthalate (CAP), are suitable for use in the preservation as well.

Formation

These carriers, and particularly proteinoids, acylated amino acids or poly amino acids, sulfonated amino acids or poly amino acids, and proteins are often insoluble or relatively insoluble in neutral or mildly acidic solutions but are also soluble, as are the imidazole derivatives useful in the present invention, in aqueous acid solutions wherein the volume to volume ratio of acid to water is greater than about 3:7. Suitable aqueous acid solvents are as above, i.e. volatile organic acids, such as for example, aqueous acetic acid, aqueous formic acid, and the like. These acids will volatilize upon nebulization or can be diluted in the aqueous solution, thereby decreasing the concentration of the acid and reversing the solubility of the carrier even in the absence of a precipitator. For example, see currently filed U.S. Pat. application Ser. No. 08/475,882, filed on Jun. 7, 1995, now U.S. Pat. No. 5,667,806, entitled “SPRAY DRYING METHOD AND APPARATUS”.

Microsphere formation occurs when the concentration of the acid in the carrier/active agent solution is decreased. As this solution is nebulized, the acid evaporates, decreasing the concentration of the acid in solution to less than 30% by volume. The carrier, then, will self assemble to form microspheres containing any optional active agent. The cargo must be stable in the concentrated acid for the time and conditions necessary to carry out the operation. Alternately, the carrier solution can be diluted, such as with water, whereby the acid concentration is decreased and the carrier precipitates to form microspheres. Preferably, the microspheres are prepared by spray drying.

The microspheres can be pH adapted by using base or acid soluble coatings including, but not limited to, proteinoid coatings, enteric coatings, acylated amino acid coatings, and the like.

Any of the solutions above may optionally contain additives such as stabilizing additives. The presence of such additives promotes the stability and dispersability of the active agent in solution. The stabilizing additives may be employed at a concentration ranging between about 0.1 and 5% (w/v), preferably about 0.5% (w/v). Suitable, but non-limiting examples of stabilizing additives include buffer salts, gum acacia, gelatin, methyl cellulose, polyethylene glycol, and polylysine.

The amount of active agent that may be incorporated in the microsphere is dependent upon a number of factors which include the concentration of active agent in the solution as well as the affinity of the active agent for the carrier. The concentration of the active agent in the final formulation also will vary depending on the required amounts for any particular end use. When necessary, the exact concentration can be determined by, for example, reverse phase HPLC analysis.

The microspheres and, therefore, the solutions described above may also include one or more enzyme inhibitors. Such enzyme inhibitors include, but are not limited to, compounds such as actinonin or epiactinonin and derivatives thereof.

The microspheres are particularly useful for administering itraconazole derivatives to any animals, including but not limited to, birds and mammals, such as primates and particularly humans; and insects. These microsphere systems are particularly advantageous for delivering these active agents as the active agent would otherwise be destroyed or rendered less effective by conditions encountered before the microsphere reaches the active agent target zone (i.e., the area in which the active agent of the delivery composition are to be released) and within the body of the animal to which they are administered. Furthermore, these microspheres can deliver relatively high amounts of the imidazole derivative and retain a high bioavailability.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples illustrate the present invention without limitation.

EXAMPLE 1

Solubilization of Itraconazole

Acetic acid solutions were prepared in water to 10%, 20%, 50% and 75% concentrations (expressed as volume glacial acetic acid/total volume of solution ×100). 100 mg itraconazole solute were then mixed independently with 1 ml of each solution and visually monitored for dissolution. If necessary, additional 1 ml aliquots of each acetic acid solution were added until the itraconazole solute was dissolved.

Results are illustrated in Table 1 below. The solubilized material did not precipitate readily.

TABLE 1 SOLUBILITY Concentration Amount of Solvent of 1 ml 1 ml 2 ml 2 ml 3 ml 3 ml 4 ml 4 ml Acid in Solvent Cold Hot Cold Hot Cold Hot Cold Hot 10% Acetic Ins. Ins. Ins. Ins. Ins. Ins. — Ins. Acid v:v 20% Acetic Ins. Ins. Ins. Ins. Ins. Part — — Acid v:v Sol. 50% Acetic Ins. Ins. Ins. Sol. Ins. — — — Acid v:v 75% Acetic Sol. — — — — — — — Acid v:v

EXAMPLE 2

Solubilization of Itraconazole

100 mg of itraconazole solute were dissolved in 1 ml glacial acetic acid solvent and aqueous acetic acid solvent at various concentrations. Results are illustrated in Table 2 below.

TABLE 2 ITRACONAZOLE SOLUTE % Acetic Acid (v:v) Dissolved Itraconazole % 100  >33 (dissolves freely on addition) 75 >10 (dissolves freely on addition) 40 5 (diss. conc. acid, then dilute) 20 2.5 (diss. conc. acid, then dilute)

EXAMPLE 3

Preparation of Itraconazole-Containing Microspheres One-Solution Method

60 grams of itraconazole solute (Janssen Pharmaceutica) were added to 1.43 liters of glacial acetic acid solvent, and the mixture was stirred to dissolve the solute. 1.43 liters of water were then added using a pump at a flow rate of 25 ml/min. Slight clouding of the solution was observed, but cleared upon further stirring. 166 grams of proteinoid (Glu-Asp-Tyr-Phe-Orn) were added and dissolved with further stirring. The final solution was filtered through folded tissue paper.

Using peristaltic pumps, the solution was fed through a Virtis SD04 spray drying apparatus under the conditions of Table 3 below.

TABLE 3 SPRAY DRYING CONDITIONS Solution flow rate 7-8 ml/min Inlet temperature 175° C. Outlet temperature 116° C. Blower speed full Compressor pressure full

Stable proteinoid microspheres containing itraconazole were formed. Analysis of typical microspheres using RP-HPLC demonstrated that they contained 14-21% itraconazole by weight.

Scanning electron microscopy in FIGS. 1A-1H illustrates that the microspheres were smooth and spherical and had diameters ranging from 0. 1μm to about 5μm. When mechanically crushed only the larger spheres shattered, while the smaller spheres remained intact. Crushing revealed a solid internal structure. See, FIG. 1G and 1H.

All patents, applications, publications, and test methods mentioned herein are hereby incorporated by reference in their entirety.

Many variations of the present invention will suggest themselves to those skilled in the art in light of the above-detailed description in which obvious variations are within the full intended scope of the appended claims. 

What is claimed is:
 1. A method for preparing imidazole derivative active agent containing microspheres, said method comprising (A) nebulizing a solution comprising (a) an imidazole active agent having the formula

 wherein R, R¹, and R² are independently hydrogen or lower alkyl; R³ is hydrogen, methyl or ethyl; R⁴ is hydrogen or methyl; Ar is phenyl, monohalophenyl, dihalophenyl, trihalophenyl, mono(lower alkyl)phenyl, di(lower alkyl)phenyl, lower alkoxyphenyl, or halothienyl; Ar¹ is phenyl, monohalophenyl, dihalophenyl, trihalophenyl, mono(lower alkyl)phenyl, di(lower alkyl)phenyl, lower alkoxyphenyl, or cyanophenyl; and n is 1 or 2; (b) a microsphere forming carrier selected from the group consisting of (i) a proteinoid; (ii) an acylated amino acid or poly amino acid or a salt thereof; (iii) a sulfonated amino acid or poly amino acid or a salt thereof; (iv) a protein or a salt thereof; (v) an enteric coating material; and (vi) any combination thereof; and (c) an active agent and carrier solubilizing effective amount of a solvent comprising an aqueous solution of at least one volatile organic acid; wherein the volume:volume ratio of acid to water in said solvent is at least about 3:7; and (B) decreasing said ratio to less than about 3:7, to yield said microspheres.
 2. A method as defined in claim 1, wherein said imidazole derivative active agent comprises itraconazole.
 3. A method as defined in claim 1, wherein said aqueous solution comprises aqueous acetic acid.
 4. A method as defined in claim 1, wherein said nebulizing is performed by spray drying.
 5. A method as defined in claim 1 wherein said ratio is decreased by volatilizing at least a portion of said acid.
 6. A method as defined in claim 1 wherein said ratio is decreased by diluting said acid.
 7. A method for preparing imidazole derivative active agent containing microspheres, said method comprising (A) nebulizing (a) solution comprising (i) an imidazole active agent having the formula

 wherein R, R¹, and R² are independently hydrogen or lower alkyl; R³ is hydrogen, methyl or ethyl; R⁴ is hydrogen or methyl; Ar is phenyl, monohalophenyl, dihalophenyl, trihalophenyl, mono(lower alkyl)phenyl, di(lower alkyl)phenyl, lower alkoxyphenyl, or halothienyl; Ar¹ is phenyl, monohalophenyl, dihalophenyl, trihalophenyl, mono(lower alkyl)phenyl, di(lower alkyl)phenyl, lower alkoxyphenyl, or cyanophenyl; and n is 1 or 2; and (ii) an active agent solubilizing effective amount of a solvent comprising an aqueous solution of at least one volatile organic acid; wherein the volume:volume ratio of acid to water in said solvent is at least about 3:7; and (b) a solution of a microsphere forming carrier selected from the group consisting of (i) a proteinoid; (ii) an acylated amino acid or poly amino acid or a salt thereof; (iii) a sulfonated amino acid or poly amino acid or a salt thereof; (iv) a protein or a salt thereof; (v) an enteric coating material; and (vi) any combination thereof; and (B) decreasing said ratio to less than about 3:7, to yield said microspheres. 